Methods suitable for the purification of particular proteins are frequently derived on a trial and error basis. Invariably, nearly all schemes developed to purify a protein to homogeneity involve a series of methods and steps which may result in yield loss and/or loss of biological activity. An objective of this invention is to provide an efficient and selective means to facilitate the purification of proteins without significant loss of yield or biological activity.
A fairly recent method for facilitating protein purification has been described by C. Bordier in The Journal of Biological Chemistry vol. 256, no. 4, pp 1604-1607 (1981) and by G. Pryde and J. Phillips in Biochem. J. vol. 233, pp 525-533 (1986). In brief, the method described in the cited references above rely on the observation that solutions of the non-ionic detergent, Triton X-114, layers into a water immiscible second phase upon warming to about 30.degree. C. The temperature at which a detergent begins forming a second, immiscible phase is defined by detergent chemists as the "cloud point." In the presence of a complex mixture of proteins, Triton X-114 may be warmed to 30-35 degrees C. whereupon hydrophilic proteins will remain with the water phase while hydrophobic proteins will migrate with the immiscible detergent phase. Use of this simple procedure to bring about separation and partial purification of proteins based on solubility and partitioning into a distinct second detergent phase has not gained widespread popularity due to the following disadvantages:
a) Due to a phenyl ring in its structure, Triton X-114 interferes with direct, spectral estimations of protein concentration.
b) Triton X-114 is not a definable, pure compound. Rather, it is a mixture containing various chain lengths of ethoxylated isooctylphenol. The use of undefined mixtures is not compatible with Good Manufacturing Practice (GMP) procedures often required for processing of pharmaceuticals.
c) Polyethoxylated nonionic detergents such as Triton X-114 frequently are contaminated or become contaminated with peroxides upon aging. This contaminant can be especially destructive to the biological integrity and activity of proteins (see H. Change and E. Bock in Anal. Biochem. vol. 104, pp 112-117 (1980).
d) In order to maintain solutions of Triton X-114 above the cloud point of 30 degrees, external heat must be continuously applied which adds mechanical complexity and further increases the risk of damaging certain sensitive proteins.